Posts Tagged ‘Regulatory T cell’

Regulatory T cells (Tregs) are important for sperm tolerance and male fertility

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Regulatory T cells (Tregs) are specialized immune cells that modulate tissue homeostasis. They are a specialized subset of T lymphocytes that function as suppressive immune cells and inhibit various elements of immune response in vitro and in vivo. While there are constraints on the number or function of Tregs which can be exploited to evoke an effective anti-tumor response, sufficient expansion of Tregs is essential for successful organ transplantation and for promoting tolerance of self and foreign antigens. Current studies have provided evidence that a defect in the number or function of Tregs contributes to the etiology of several reproductive diseases.

In the male reproductive tract, prevention of autoimmune responses against antigenic spermatozoa, while ensuring protection against stressors, is a key determinant of fertility. Using an autoimmunity-induced model, it was uncovered that the role of Tregs in maintaining the tolerogenic state of the testis and epididymis. The loss of tolerance induced an exacerbated immune cell infiltration and the development of anti-sperm antibodies, which caused severe male subfertility. By identifying immunoregulatory mechanisms in the testis and epididymis.

Tregs modulate tissue homeostatic processes and immune responses. Understanding tissue-Treg biology will contribute to developing precision-targeting treatment strategies. Here, it was reported that Tregs maintain the tolerogenic state of the testis and epididymis, where sperm are produced and mature. It was found that Treg depletion induces severe autoimmune orchitis and epididymitis, manifested by an exacerbated immune cell infiltration [CD4 T cells, monocytes, and mononuclear phagocytes (MPs)] and the development of anti-sperm antibodies (ASA).

In Treg-depleted mice, MPs increased projections toward the epididymal lumen as well as invading the lumen. ASA-bound sperm enhance sperm agglutination and might facilitate sperm phagocytosis. Tolerance breakdown impaired epididymal epithelial function and altered extracellular vesicle cargo, both of which play crucial roles in the acquisition of sperm fertilizing ability and subsequent embryo development. The affected mice had reduced sperm number and motility and severe fertility defects.

Deciphering these immunoregulatory mechanisms may lead to the development of therapies for infertility and identifying potential targets for immuno-contraception. Ultimately, such knowledge fills gaps related to reproductive mucosa, which is an understudied facet of human male health.







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Highlighted Progress in Science – 2017

Reporter: Sudipta Saha, PhD


  1. Lungs can supply blood stem cells and also produce platelets: Lungs, known primarily for breathing, play a previously unrecognized role in blood production, with more than half of the platelets in a mouse’s circulation produced there. Furthermore, a previously unknown pool of blood stem cells has been identified that is capable of restoring blood production when bone marrow stem cells are depleted.


  1. A new drug for multiple sclerosis: A new multiple sclerosis (MS) drug, which grew out of the work of UCSF (University of California, San Francisco) neurologist was approved by the FDA. Ocrelizumab, the first drug to reflect current scientific understanding of MS, was approved to treat both relapsing-remitting MS and primary progressive MS.


  1. Marijuana legalized – research needed on therapeutic possibilities and negative effects: Recreational marijuana will be legal in California starting in January, and that has brought a renewed urgency to seek out more information on the drug’s health effects, both positive and negative. UCSF scientists recognize marijuana’s contradictory status: the drug has proven therapeutic uses, but it can also lead to tremendous public health problems.


  1. Source of autism discovered: In a finding that could help unlock the fundamental mysteries about how events early in brain development lead to autism, researchers traced how distinct sets of genetic defects in a single neuronal protein can lead to either epilepsy in infancy or to autism spectrum disorders in predictable ways.


  1. Protein found in diet responsible for inflammation in brain: Ketogenic diets, characterized by extreme low-carbohydrate, high-fat regimens are known to benefit people with epilepsy and other neurological illnesses by lowering inflammation in the brain. UCSF researchers discovered the previously undiscovered mechanism by which a low-carbohydrate diet reduces inflammation in the brain. Importantly, the team identified a pivotal protein that links the diet to inflammatory genes, which, if blocked, could mirror the anti-inflammatory effects of ketogenic diets.


  1. Learning and memory failure due to brain injury is now restorable by drug: In a finding that holds promise for treating people with traumatic brain injury, an experimental drug, ISRIB (integrated stress response inhibitor), completely reversed severe learning and memory impairments caused by traumatic brain injury in mice. The groundbreaking finding revealed that the drug fully restored the ability to learn and remember in the brain-injured mice even when the animals were initially treated as long as a month after injury.


  1. Regulatory T cells induce stem cells for promoting hair growth: In a finding that could impact baldness, researchers found that regulatory T cells, a type of immune cell generally associated with controlling inflammation, directly trigger stem cells in the skin to promote healthy hair growth. An experiment with mice revealed that without these immune cells as partners, stem cells cannot regenerate hair follicles, leading to baldness.


  1. More intake of good fat is also bad: Liberal consumption of good fat (monounsaturated fat) – found in olive oil and avocados – may lead to fatty liver disease, a risk factor for metabolic disorders like type 2 diabetes and hypertension. Eating the fat in combination with high starch content was found to cause the most severe fatty liver disease in mice.


  1. Chemical toxicity in almost every daily use products: Unregulated chemicals are increasingly prevalent in products people use every day, and that rise matches a concurrent rise in health conditions like cancers and childhood diseases, Thus, researcher in UCSF is working to understand the environment’s role – including exposure to chemicals – in health conditions.


  1. Cytomegalovirus found as common factor for diabetes and heart disease in young women: Cytomegalovirus is associated with risk factors for type 2 diabetes and heart disease in women younger than 50. Women of normal weight who were infected with the typically asymptomatic cytomegalovirus, or CMV, were more likely to have metabolic syndrome. Surprisingly, the reverse was found in those with extreme obesity.


























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Reporter: Aviva Lev-Ari, PhD, RN




Trypanosoma cruzi Trans-Sialidase Initiates a Program Independent of the Transcription Factors RORγt and Ahr that Leads to IL-17 Production by Activated B Cells

Researchers identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17+ B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. [Nat Immunol] Abstract

PUBLICATIONS (Ranked by impact factor of the journal)

Autoimmune Pancreatitis in MRL/Mp Mice Is a T Cell-Mediated Disease Responsive to Cyclosporine A and Rapamycin Treatment

The authors report that blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell function and raised the effector T cell response with subsequent histomorphological organ destruction, indicating that autoimmune pancreatitis is a T cell-driven disease. Using an established histopathological score, they found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. [Gut] Abstract

Excessive Th1 Responses Due to the Absence of TGF-β Signaling Cause Autoimmune Diabetes and Dysregulated Treg Cell Homeostasis

TGF-β signaling in T cells is critical for peripheral T-cell tolerance by regulating effector CD4+ T helper (Th) cell differentiation. However, it is still controversial to what extent TGF-β signaling in Foxp3+ regulatory T (Treg) cells contributes to immune homeostasis. Researchers showed that abrogation of TGF-β signaling in thymic T cells led to rapid type 1 diabetes development in NOD mice transgenic for the BDC2.5 T-cell receptor. [Proc Natl Acad Sci USA] Abstract

Inhibition of PDE4B Suppresses Inflammation by Increasing Expression of the Deubiquitinase CYLD

The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defense response. Researchers showed that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. [Nat Commun] Abstract | Press Release

Regulatory T Cells Prevent Plaque Disruption in Apolipoprotein E-Knockout Mice

Investigators report that adoptive transfer of regulatory T cells dose-dependently changed plaque composition to a stable plaque phenotype and lowered the incidence of plaque disruption in apolipoprotein E-knockout mice. The major mechanisms involved reduced expression of inflammatory cytokines and matrix metalloproteinase (MMP)-2 and MMP-9, and enhanced expression of P4Hα1 in the carotid plaque. [Int J Cardiol] Abstract

GARP-TGF-β Complexes Negatively Regulate Regulatory T Cell Development and Maintenance of Peripheral CD4+ T Cells In Vivo

Researchers address the role of Glycoprotein A Repetitions Predominant (GARP) in regulating regulatory T cells and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. They found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. [J Immunol] Abstract

Fine-Tuning of Regulatory T Cell Function: The Role of Calcium Signals and Naive Regulatory T Cells for Regulatory T Cell Deficiency in Multiple Sclerosis

As regulatory T cells (Tregs) counteract the sustained elevation of intracellular calcium, which is indispensable for full activation of conventional T cells (Tcons), the authors hypothesized that interference with this pathway might prompt multiple sclerosis-related Treg dysfunction. Using single-cell live imaging, they observed that Tregs rapidly reduce Ca2+ influx and downstream signals in Tcons upon cell contact, yet differ in their potency to efficiently suppress several target cells at the same time. [J Immunol] Abstract

Systems Model of T Cell Receptor Proximal Signaling Reveals Emergent Ultrasensitivity

Researchers used a systems model to show that signaling architecture produces emergent ultrasensitivity resulting in switch-like responses at the scale of individual T cell antigen receptors. Importantly, this switch-like response is an emergent property, so that removal of multiple immunoreceptor tyrosine-based activation motifs, sequential phosphorylation, or differential affinities abolishes the switch. [PLoS Comput Biol]

Full Article

Decreased Regulatory T-Cells and CD4+/CD8+ Ratio Correlate with Disease Onset and Progression in Patients with Generalized Vitiligo

Scientists evaluated CD4+/CD8+ ratio and CD4+CD25hiFoxP3+ regulatory T cells (Tregs) in Generalized Vitiligo (GV) patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4+/CD8+ ratio and Tregs in 82 patients and 50 controls. CD8+ T-cell counts were significantly higher in GV patients as compared to controls. [Pigment Cell Melanoma Res] Abstract

Sex Bias in Experimental Immune-Mediated, Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs, Estrogen, and IL-6

Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, researchers investigated their role in a mouse model of immune-mediated drug-induced liver injury. [PLoS One] Full Article

The Hedgehog Receptor Patched1 in T Cells Is Dispensable for Adaptive Immunity in Mice

Hedgehog (Hh) signaling modulates T cell development and function but its exact role remains a matter of debate. To further address this issue, researchers made use of conditional knock-out mice in which the Hh receptor Patched1 is inactivated in the T cell lineage. [PLoS One] Full Article



Modulating T Regulatory Cells in Cancer: How Close Are We?

The authors provide a historical perspective of the discovery of regulatory T cells (Tregs), followed by a summary of the existing literature on the role of Tregs in malignancy. [Immunol Cell Biol] Abstract

Importance of Reverse Signaling of the TNF Superfamily in Immune Regulation

F-related ligands (with the exception of lymphotoxin-α) are synthesized as type II transmembrane proteins, though many of them also have soluble forms. An increasing number of publications report that these ‘ligands’ behave as receptors, activating intracellular signaling pathways when interacting with cognate ‘receptors’ or agonistic antibodies. [Expert Rev Clin Immunol] Abstract

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Immunotherapies against Cancer: German Cancer Research Center to Broaden Strategic Alliance with Bayer HealthCare

The German Cancer Research Center and Bayer HealthCare will extend their successful strategic research alliance in search of novel cancer therapeutics by focusing their activities also on the field of immunotherapy. [German Cancer Research Center] Press Release

Pitt Team Gets $5 Million National Institutes of Health Grant to Make Vaccine Component that Stimulates TB-Fighting T-Cells

Researchers at the University of Pittsburgh School of Medicine have received a $5 million federal grant to develop a vaccine ingredient that can generate the type of immune response needed to protect against tuberculosis (TB) infection. [UPMC/University of Pittsburgh Schools of the Health Sciences] Press Release

T Cell Biology Pioneer Allison Wins First AACR Honor for Cancer Immunology

The scientist whose discoveries led to the first drug approved for metastatic melanoma by “treating the immune system, not the cancer,” also is the first to receive the AACR-CRI Lloyd J. Old Award in Cancer Immunology. James Allison, Ph.D., professor and chair of The University of Texas MD Anderson Cancer Center Department of Immunology, was honored at the AACR Annual Meeting 2013 in Washington, D.C. [The University of Texas MD Anderson Cancer Center] Press Release

Immune Therapy from Austria Receives Orphan Drug Designation from European Medicines Agency

The European Medicines Agency recently awarded the Austrian biotech company Activartis an Orphan Drug Designation for its innovative Cancer Immune Therapy AV0113. The Orphan Drug Designation applies specifically to the use of AV0113 for the treatment of glioma, a type of brain tumor, which afflicts around one in 10.000 people in the EU. [Activartis Biotech GmbH] Press Release

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IRF-1 Deficiency Skews the Differentiation of Dendritic Cells

Reporter: Larry H Bernstein, MD, FCAP



IFN Regulatory Factor-1 Negatively Regulates CD4+CD25+ Regulatory T Cell Differentiation by Repressing Foxp3 Expression1


Alessandra Fragale*, Lucia Gabriele†, Emilia Stellacci*, Paola Borghi†,…. and Angela Battistini2,*
The Journal of Immunology   Aug 1, 2008; 181(3): 1673-1682

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance,

  • mechanisms and molecules involved in the generation of Treg cells remain poorly understood.

IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell

  • development and function by specifically repressing Foxp3 expression.

IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore,

  • IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas
  • restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells
    • impaired their differentiation into CD25+Foxp3+ cells.

Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice

  • exhibited more increased suppressive activity than wild-type Treg cells.

Such phenotype and functional characteristics were explained at a mechanistic level by the finding that

  • IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and
  • negatively regulates its transcriptional activity.

We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells

  • through direct repression of Foxp3 expression.

Tolerance is critical for prevention of autoimmunity and maintenance of immune homeostasis by active suppression of inappropriate immune responses. Suppression has a dedicated population of  T cells that

  • control the responses of other T cells.

This cell population, referred to as regulatory T (Treg)3 cells, actually comprises several subsets, including naturally occurring CD4+CD25+ Treg cells that arise in thymus. Once generated,

  • thymic Treg cells are exported to peripheral tissues, and
  • comprise 5–10% of peripheral CD4+ T cells (1, 2, 3).

CD4+CD25+ Treg cells are characterized by

  • constitutive expression of IL-2Rα (CD25), CTLA-4, and glucocorticoid-induced TNFR family-related gene; moreover,
  • they express CD62 ligand (CD62L) and are mainly CD45RBlow (4).

In contrast to cell surface markers, which can be shared with other T cells populations,

  • the forkhead/winged-helix family transcriptional repressor Foxp3 is
  • specifically expressed in CD4+CD25+ Treg cells and
  • rigorously controls their development and function (5, 6, 7).

Functionally after TCR stimulation, CD4+CD25+ Treg cells can

  • mediate strong suppression of proliferation and
  • IL-2 production by CD4+ T cells both in vivo and in vitro (8).

Although mechanisms of suppression are not fully understood,

  • they appear to be cell contact-mediated, whereas
  • the relative contribution of soluble cytokines remains controversial
    • with differences between in vitro and in vivo results (1, 8, 9).

Indeed, the involvement of cytokines in the suppressor function of CD4+CD25+ Treg cells has been proposed in vivo,

  • where they are able to produce IL-10 and TGF-β (10, 11, 12), and
  • importantly, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells (13).

Beside naturally occurring CD4+CD25+ Treg cells, CD4+CD25+ Treg cells can also be

  • induced (inTreg) in vivo or in vitro after TCR stimulation and TGF-β treatment,
  • acquiring expression of CD25 and Foxp3 both in mice (14, 15, 16) and humans (17, 18, 19, 20),
    • although with characteristic functional differences (20).

Despite extensive studies on the role of Foxp3 in inducing and maintaining tolerance, little information on regulation of its expression is available. Transcription factors of the IFN regulatory factor (IRF) family participate in

  • the early host response to pathogens,
  • in immunomodulation and
  • hematopoietic differentiation (21).

Nine members of this family have been identified based on a unique helix-turn-helix DNA binding domain, located at

  • the N terminus that is responsible for binding to the IRF consensus element (IRF-E) (21).
The first member of the family, IRF-1, was originally identified as a protein that binds
  • the cis-acting DNA elements in the ifnβ gene promoter and the IRF-E (also referred to as the IFN-stimulated response element; ISRE),
  • in the promoters of IFN-αβ-stimulated genes (22).

IRF-1 is expressed at low basal levels in all cell types examined, but

  • accumulates in response to several stimuli and cytokines including IFN-γ, the strongest IRF-1 inducer (22).
Intensive functional analyses conducted on this transcription factor have revealed a remarkable functional diversity in the
  • regulation of cellular responses through the
  • modulation of different sets of genes,
  • depending on
    1. cell type,
    2. state of the cell, and/or
    3. nature of the stimuli (21).
We and others have shown that IRF-1 affects the differentiation of both lymphoid and myeloid lineages (22, 23, 24, 25, 26, 27, 28). In particular, studies in knockout (KO) mice have implicated IRF-1
in the regulation of various immune processes:
  1. impairment of CD8+ T cell and NK cell maturation,
  2. impaired IL-12 macrophage production,
  3. exclusive Th2 differentiation, and
  4. defective Th1 responses…………. have all been observed (22, 23, 24, 25, 26).
As a result, IRF-1−/− mice are highly susceptible to infections, for which effective host control
    • is associated with a Th1 immune response (24).
In contrast, these mice are characterized by
  • increased resistance to several autoimmune diseases such as
  1. collagen-induced arthritis,
  2. experimental autoimmune encephalomyelitis,
  3. Helicobacter pylori-induced gastritis,
  4. induced lymphocytic thyroiditis,
  5. insulitis, or
  6. diabetes (29, 30, 31, 32).
Recently, we reported that IRF-1−/− mice display a prevalence of
  • dendritic cell (DC) subsets with immature and tolerogenic features that were
    • unable to undergo full maturation after stimulation.
Moreover, IRF-1−/− DC conferred
    • increased suppressive activity to CD4+CD25+ Treg cells (33).
Because there is growing evidence that immature or partially matured DC can induce tolerance (34, 35), we hypothesized that IRF-1 could play a role in
  • Treg development and function.
In this study, we analyzed the CD4+CD25+ compartment in IRF-1−/− mice and
  • we found that in vivo IRF-1 deficiency resulted in a
  • selective and marked increase in highly differentiated and activated CD4+CD25+Foxp3+ Treg cells, whereas
reintroduction of IRF-1 by retrovirus transduction
    • impaired TGF-β-mediated differentiation of IRF-1−/− CD4+CD25− T cells into CD4+CD25+Foxp3+ Treg cells.
At molecular level, we show that IRF-1 plays a direct role in the generation and expansion of CD4+CD25+ Treg cells
    • specifically repressing Foxp3 transcriptional activity.
Our results, therefore, highlight a unique role for IRF-1 as regulator of Foxp3, thus pointing to IRF-1 as a specific tool to control altered tolerance.
CD4+CD25+ Treg from IRF-1−/− mice are increased and functionally more suppressive than WT Treg cells
The distribution and the phenotype of CD4+CD25+Foxp3+ Treg in lymphoid organs of IRF-1−/− mice were determined by flow cytometry.
the number of ex vivo double positive CD4+CD25+ cells was significantly increased in spleens and skin draining and mesenteric lymph nodes (2.8-, 2.3-, and 2.1-fold increase, respectively), and to a lesser extent, in thymus (1.6-fold increase) of IRF-1−/− mice as compared with WT mice. Consistently with previous reports (23, 41), no differences in CD4+ T cell and total cell numbers in all lymphoid organs from WT or IRF-1−/− mice were found (data not shown). Strikingly, intracellular analysis of Foxp3 expression showed that this factor was increasingly expressed in CD4+CD25+ Treg cells from spleens as well as from other lymphoid organs of IRF-1−/− mice
FACS analysis of splenic magnetically sorted CD4+CD25+ Treg cells was performed to evaluate the expression of activation markers.  IRF-1−/− Treg cells were to a large extent characteristic of a marked activated and differentiated phenotype.
Because there is accumulating evidence that activity of CD4+CD25+ Treg cells in vivo involves some immunosuppressive cytokines (9, 10, 11, 12), we also compared the cytokine profile of IRF-1−/− CD4+CD25+ Treg cells with the profile of WT counterparts . Lower levels of proinflammatory cytokines, such as TNF-α and IFN-γ, whereas higher levels of IL-4 were expressed in CD4+CD25+ Treg cells as well as in CD4+CD25− T lymphocytes from KO as compared with WT cells. Notably, only IRF-1−/− Treg cells showed a clear-cut increase in the expression of IL-10. By contrast, TGF-β was expressed at similar levels in CD4+CD25+ Treg cells from both IRF-1−/− and WT mice. Accordingly with mRNA data, IL-10 secretion in supernatants of TCR-stimulated CD4+CD25+ cocultures from IRF-1−/− mice was significantly increased (3-fold), whereas
    • IFN-γ secretion was decreased (2.5-fold) compared with cocultures from WT mice (Fig. 2⇑C).
As the functional hallmark of Treg cells is their ability to suppress the expansion of effector T cells, we next evaluated this activity performing suppression assays (1, 2, 3, 8). Importantly, CD4+CD25+ Treg cells from IRF-1−/− mice were found significantly more efficient than WT Treg cells in suppressing the proliferation of syngeneic CD4+CD25− responder T cells in a dose-dependent fashion. Next, to verify whether IRF-1−/− Treg cells suppression ability was retained vs WT responder T cells, we performed suppression assays using IRF-1−/− Treg and WT responders and vice versa. The suppressive activity of IRF-1−/− Treg cells toward WT responders was dose-dependently increased, as well.
IRF-1−/− CD4+CD25− T cells show high bent to convert into CD4+CD25+ Treg cells
It has been reported in mice and human that TGF-β promotes the induction of peripheral CD4+CD25− T cells into CD4+CD25+ Treg cells (inTreg), that acquire Foxp3 expression and regulatory functions.
In presence of TGF-β, 44.2% of CD4+CD25+ inTreg cells were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, whereas
  • only 24% of double positive cells were detected in the corresponding coculture from WT mice.
Notably, even in absence of TGF-β, 25.4% CD4+CD25+ inTreg were generated in the coculture of CD4+CD25− T cells from IRF-1−/− mice, as
  • compared with 16.5% of Treg cells generated in WT cocultures.
Importantly, an increased number of CD4+CD25+-gated Foxp3+ cells were observed in IRF-1−/− inTreg cells in the presence (4.5-fold increase) or in the absence (8-fold increase) of TGF-β compared with WT inTreg cells. Next, to evaluate quantitatively Foxp3 expression levels in TGF-β-induced Treg vs ex vivo freshly purified Treg cells, quantitative real-time PCR was performed. A clear-cut
induction of Foxp3 mRNA (4.5-fold increase) was detected in TGF-β-treated IRF-1−/− cells compared with WT cells. Of note, these levels were comparable with those present in freshly isolated IRF-1−/− CD4+CD25+ cells. Strikingly, also untreated IRF-1−/− T cells showed higher levels of Foxp3 mRNA than WT untreated cells (6-fold increase) and similar to levels present in freshly purified WT CD4+CD25+ Treg cells.
The functionality of CD4+CD25+Foxp3+ inTreg cells was then assessed by suppression assays. TGF-β-treated IRF-1−/− inTreg cells were significantly more effective than the WT counterpart cells
  • in suppressing proliferation of effector T cells in a dose-dependent way.
Interestingly, a saturating amount of anti-IL-10 m Abs neutralized the suppression ability of  inTreg cells from both IRF-1−/− and WT mice even though the effect was much more marked in IRF-1−/− inTreg cells. Control Abs did not exhibit any effect.
Restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD4+CD25+Foxp3+ cells
To address the specificity of IRF-1 role in differentiation of CD4+CD25+ Treg cells from CD25− cells, we investigate whether
  • forced expression of IRF-1 in CD4+CD25− IRF-1−/− T cells could rescue the WT phenotype.
  • bicistronic retroviral vectors expressing murine IRF-1 and human CD8 protein as surface marker (MigR1 IRF-1-CD8) or CD8 alone (MigR1 EV-CD8) were generated.
Splenic CD4+CD25− cells from IRF-1−/− mice were stimulated with plate-bound anti-CD3 and anti-CD28 Abs and infected with either retrovirus.
  • 31.6% of MigR1 EV-CD8 CD4+ retrovirus-infected cells were CD25+, by contrast
  • only 17.7% of MigR1 IRF-1-CD8 retrovirus-infected cells were double positive.
Consistently, Foxp3 expression in CD8+-gated cells was significantly decreased in MigR1 IRF-1-CD8-infected cells as compared with
  • those infected with MigR1 EV-CD8 vectors,
  • strongly supporting the evidence that IRF-1 specifically impairs CD4+CD25+ cell differentiation.
IRF-1 binds an IRF-E on the Foxp3 core promoter and inhibits its transcriptional activity
To shed light on the molecular mechanisms responsible for the striking effect exerted by IRF-1 on the development and function of CD4+CD25+ Treg cells, we investigated whether IRF-1, which is a regulator of key immunomodulatory genes (21), could directly regulate the foxp3 gene promoter activity. The proximal promoter of human foxp3 gene has been recently characterized and localized at −511/+176 bp upstream of the 5′ untranslated region (38). By the Genomatix software, we analyzed this region and found an IRF-E spanning from −234 to −203 bp . This region has been found highly homologous to mouse and rat foxp3 promoter, and of note, the IRF-E is perfectly conserved between humans and these species (38). To determine whether IRF-1 could bind this sequence, DNA affinity purification assays were performed with cell extracts from Jurkat T cells, which display discrete basal levels of IRF-1, and from the same cells treated with IFN-γ to maximally stimulate IRF-1 expression. A total of 200 μg of nuclear extracts was incubated with oligonucleotides containing the WT or the a mutated version of IRF-E. The isolated complexes were then examined by immunoblotting against IRF-1. A specific binding of IRF-1 to Foxp3 oligonucleotide was evident. The binding was strongly stimulated by IFN-γ treatment and, interestingly, it was comparable to that obtained when the same extracts were incubated with a synthetic oligonucleotide corresponding to C13, the canonical IRF-1 consensus sequence (21). IRF-1 binding was highly specific because a mutated version of the Foxp3/IRF-E, or an unrelated oligonucleotide corresponding to the STAT binding site present on the β-casein gene promoter, did not retain any protein from the same extracts. To functionally characterize the specific binding of IRF-1 to the foxp3 gene promoter, we cloned the encompassing part of the proximal promoter containing the IRF-E from −296 to +7 bp of foxp3 gene promoter upstream the luciferase reporter gene. The effect of IRF-1 was evaluated in Jurkat T cells transiently cotransfected with the luciferase reporter gene and increasing doses of an IRF-1-expressing vector.
The results indicated that the basal transcriptional activity of the foxp3 gene promoter
    • was substantially reduced in the presence of IRF-1 and the effect was dose-dependent.
Conversely, the basal activity of the foxp3 gene promoter construct mutated in the IRF-E
    • was not affected by IRF-1 overexpression.
Interestingly, IRF-2, a repressor of IRF-1 transcriptional activity on most promoters (21), neither affected the promoter activity nor counteracted the inhibitory effect exerted by IRF-1.  IRF-1, IRF-2, as well as the IFN-γ treatment drastically reduced the transcriptional activity of the il4 gene promoter, whereas
  • the low molecular mass polypeptide lmp2 construct was stimulated by IRF-1 and by IFN-γ treatment, but it was not affected by IRF-2.

All together these results demonstrate the specificity and functional relevance of IRF-1 binding to the foxp3 proximal promoter.

Foxp3 is a direct target of IRF-1 in human and mouse primary CD4+CD25− T cells and CD4+CD25+ Treg cells
To assess the biological relevance of the the reported effects of IRF-1 on Treg development and on the regulation of Foxp3 expression, we performed experiments with primary cells. We first assessed by Western blot IRF-1 expression levels in CD4+CD25+ Treg cells vs CD4+CD25− T cells magnetically sorted from PBMC of healthy donors or from mice spleens. Strikingly, we found that IRF-1 was down-regulated in double positive cells as compared with CD4+CD25− T cells both in mouse and human primary cells. To determine whether IRF-1 binds the Foxp3 oligonucleotides in primary Treg cells, pull-down assays with the same extracts were then performed. IRF-1 binding to Foxp3 oligonucleotide was significantly decreased in primary CD4+CD25+ Treg cells compared with CD4+CD25− T cells from both species. Foxp3 staining of CD4+CD25− T cells and CD4+CD25+ human Treg cells confirmed that these cells expressed low and high levels of Foxp3, respectively, and
  • Foxp3 expression was further increased by IL-2 treatment.
To test whether IRF-1 expression was also down-modulated during the acquisition of Treg cell phenotype upon TGF-β treatment, freshly purified TCR-activated CD4+CD25− T cells from both species were cultured with TGF-β, or left untreated, for 3 days and Western blot analysis was performed. When cells were cultured in presence of TGF-β, IRF-1 expression was substantially decreased, as compared with untreated cells. Pull-down assays revealed that IRF-1 binding to Foxp3 oligonucleotide was decreased in TGF-β-treated primary cells compared with untreated cells, as well. Consistently, FACS analysis of these cultures indicated that ∼35% of TGF-β-treated CD4+ cells were Foxp3+ in human and ∼10% in mouse TGF-β treated cultures, respectively. By contrast, even though 46.3% of human untreated cells were CD25+ only 5% were Foxp3+.
Next, we assessed the in vivo IRF-1 binding to foxp3 gene in human and mouse primary magnetically sorted CD4+CD25− T cells and CD4+CD25+ Treg cells, using ChIP assay with anti-IRF-1 Abs. After DNA immunoprecipitation, subsequent real-time PCR amplification of the foxp3 gene surrounding the IRF-E site showed significant IRF-1 binding to Foxp3 promoter in CD4+CD25−Foxp3− T cells, and by contrast, a 5-fold decrease of IRF-1 binding in CD4+CD25+Foxp3high human Treg cells (Fig. 6⇑C). Similarly, the binding of IRF-1 to the Foxp3 promoter in the mouse Treg cells was decreased by ∼50%.
Finally, to assess the functionality of the in vivo IRF-1 binding, negatively selected primary human and mouse CD4+ T lymphocytes were nucleofected with the Foxp3 luciferase reporter gene along with expression vector for IRF-1. Fig. 6⇑E shows the results obtained with T cells from three different healthy donors and Fig. 6⇑F shows a representative experiment with mouse T cells from three independent experiments. In all samples, a discrete basal activity of foxp3 gene promoter was present and this activity was significantly repressed by IRF-1.
The identification of molecules controlling Treg differentiation and function is important not only in understanding host immune responses in malignancy and autoimmunity but also in shaping immune response.
In this study, we have shown that IRF-1, a transcription factor involved in the IFN signaling, selectively affects CD4+CD25+ Treg cell development and function, unraveling a novel immunoregulatory function of IRF-1 in addition to its well-established role in balancing Th1 vs Th2 type immune responses. Several lines of evidence support this conclusion:
1) IRF-1−/− mice show a selective and marked increase in all lymphoid organs of CD4+CD25+Foxp3+ Treg cells; 2) CD4+CD25+ from IRF-1−/− mice are characterized by a highly activated and differentiated  phenotype and higher levels of Foxp3 that make them to be functionally more suppressive than WT Treg cells;
3) after TGF-β treatment, and importantly also in its absence, CD4+CD25− T cells from KO mice promptly converted into CD4+CD25+Foxp3+ Treg with a higher suppressive activity than WT cells;
4) forced retrovirus-mediated expression of IRF-1 in IRF-1−/− CD4+CD25− T cells impairs their differentiation into CD25+Foxp3+ cells; and 5) IRF-1 directly regulates transcriptional activity of the foxp3 gene promoter.
The phenotypical and functional characteristics of IRF-1−/− Treg cells strongly support the conclusion that IRF-1 can be considered a key negative regulator of CD4+CD25+ Treg cells.
The increased frequency of differentiated and activated CD4+CD25+ Treg cells characterized by an immunosuppressive cytokine profile described in this study
    • may provide a mechanistic base for the reduced incidence and severity of several autoimmune diseases characterizing IRF-1−/− mice .
In this regard, it has been recently shown that CD4+CD25+ Treg cells were increased in IRF-1−/− mice backcrossed with the MRL/lpr mice, which showed reduced glomerulonephritis.
The increased production of the immunosuppressive cytokine IL-10 by isolated Treg cells from IRF-1−/− mice and the reverted suppression ability of inTreg by anti-IL-10 Abs suggest that this cytokine could play a key role in their suppressor function. Consistently, IL-10 activity has been recently associated with the function of TGF-β-induced CD4+CD25−CD45RBlow cells because their suppressive activity was abrogated with anti-IL-10R Ab treatment (13). Moreover, several reports focused on the in vivo IL-10 role in peripheral CD4+CD25+ Treg cell function in various autoimmunity models (10, 11, 12), although IL-10 seems not required for the functions of thymically derived Treg cells (1). In contrast with the increased IL-10 production, T cells from IRF-1−/− mice failed to produce significant amounts of proinflammatory cytokines such as IFN-γ or TNF-α. Accordingly, an inverse relationship between in vivo IFN-γ administration and generation or activation of CD4+CD25+ Treg cells has been recently shown (45). Moreover, in humans, it has been reported that TNF-α inhibits the suppressive function of both naturally occurring CD4+CD25+ Treg and TGF-β-induced Treg cells, and an anti-TNF Ab therapy reversed their suppressive activity by down-modulating the expression of Foxp3 (46). These latter and our results are apparently in contrast with what was recently reported on the stimulating role of IFN-γ on Foxp3 induction and conversion of CD4+CD25− T cells to CD4+ Treg cells in the IFN-γ KO model (47). In this regard, it is noteworthy to underline that, as it has been also suggested, although knocking down genes involved in up-regulation of IFN-γ expression do not significantly influence autoimmunity, by contrast the absence of genes expressed in response to IFN-γ, including IRF-1, lead to greatly reduced autoimmunity (48). Thus, although the exact mechanism underlying IFN-γ and TNF-α interference with the elicitation of Treg cells remains to be defined, we can speculate that induction of IRF-1 expression, which is up-regulated by IFN-γ and TNF-α, may represent a mechanism through which proinflammatory cytokines negatively affect Foxp3 expression, thereby influencing generation or activation of CD4+CD25+ Treg cells.
It is well known that Foxp3 plays a pivotal role in the regulatory functions of CD4+CD25+ T cells both in humans and in animal models. Thus, the key question in the field of Treg biology is which are molecules and signals that govern Foxp3 transcription.
We identify Foxp3 as specific target of IRF-1 and we show
    • that it binds to foxp3 gene promoter in vitro and in vivo and represses its expression.
Structure of the human foxp3 gene promoter and elements necessary for its induction in T cells have been reported. We have identified an IRF-E sequence at 203 bp upstream of the transcriptional start site that is highly conserved. This element is bound by IRF-1 as proven by pull-down experiments and by ChIP analysis in intact cells, and IRF-1 binding resulted in a specific,
  • dose-dependent repression of the foxp3 proximal promoter.
Notably, treatments with IFN-γ, a major IRF-1 inducer, significantly inhibited foxp3 gene promoter transcriptional activity, whereas IRF-2 did not have any effects. It is noteworthy that the foxp3 gene is highly conserved between mouse and man species, and in particular, the core promoter and the IRF-E identified in this study are perfectly conserved between mouse and human. Such conservation underscores the importance of this motif as regulatory element and provides additional evidence for the role of IRF-1 in regulating foxp3 gene expression.  IRF-1 binds this sequence and negatively regulates its expression in both human and mouse cells. The molecular interactions enabling IRF-1 to inhibit Foxp3 are not yet identified, although our preliminary results show that IRF-1 may compete with c-Myb for the binding to the same overlapping consensus sequence on the foxp3 gene promoter.
In summary, the current study provides evidence that IRF-1 affects CD4+CD25+ development and function by Foxp3 repression. Thus, our data demonstrate a new important contribution by which IRF-1 affects T cell differentiation and provide new important insights into molecular mechanisms controlling immune homeostasis.

Th1-Th2-Th17-Treg origin

Th1-Th2-Th17-Treg origin (Photo credit: Wikipedia)


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